Method and apparatus for the preparation of monolayers of particles or molecules

ABSTRACT

A method and an apparatus are described to prepare monolayers of particles (or molecules ) ( 3 ), realizing the steps of: injecting a liquid film ( 2 ) with particles ( 3 ) dispersed on the external surface of a rotary member ( 1 ); adjusting the surface charge density of the particles ( 3 ) by injecting adsorption reagents ( 10 ), carrying particles ( 3 ) placed on film ( 2 ) surface so that they form a substantially uniform monolayer ( 5 ); putting the monolayer ( 5 ) on the liquid film surface ( 2 ) in contact with a substrate ( 7 ); and advancing the rotary member ( 1 ) longitudinally with respect to the substrate ( 7 ), in both directions, so that the monolayer ( 5 ) is detached from the liquid film ( 2 ) and attached to the substrate ( 7 ).

[0001] The present invention refers to a method and a apparatus for thepreparation of monolayers of particles or molecules.

[0002] The fabrication of monolayers of insoluble particles to thegas-liquid interface was realized through uses of troughs usually fullof aqueous solutions. To the gas-water interface, solutions containingamphiphilic molecules are usually spreaded, these being molecules madeof a polar head and a chain of fatty acids. After the volatile solventhas evaporated, it leaves at the gas-liquid interface the amphiphilicmolecules. Finally, a mobile barrier compresses the molecules in amonolayer. Therefore, essentially there occurs an immobile troughcontaining an unmoving subphase or which molecules are laterallytransported through it by exploiting the surface tension differencebetween the subphase and the deposited solution, and a mobile barrier.

[0003] The transfer of the monolayer onto a solid substrate is realizedthrough several methods. One is the so-called Langmuir-Blodgett method,and essentially comprises a vertical immersion of a solid plate in thesubphase through the monolayer; by pulling up such plate, the layer istransferred onto the plate by lateral compression. That can be repeatedmany times. Another method, called the Langmuir-Schaeffer method,comprises the descent of an horizontal plate onto the monolayer. After acontact is made, the plate is again extracted with the monolayer on it.

[0004] In order to improve the fabrication of insoluble particles,several attempts have been carried out. One has been to make a cylinderrotate under the water surface. One expected that such movement drovethe insoluble particles ahead in a forming monolayer. However, in themajority of cases, this technique requires a precompression of analready prepared monolayer. The cylinder that compresses the layer ismade of hydrophobic material. Moreover, only insoluble molecules areusable. Another device has been recently disclosed by G. Fuller, C.Franck and C. Robertson (Langmuir, 10, 1251 (1994)). It comprises thecompression of insoluble particles with a flowing subphase between afixed surface and the monolayer surfaces. Again, only insolublemolecules are used.

[0005] There are several limits in these previous methods: the essentialone is that these methods are provided for insoluble particles. Theattempts to extend the above methods to soluble particles have suppliedmarginal results. Slowness, loss of particles, low reproducibility anddenaturation of proteins are a general characteristic of these methods.

[0006] The Applicant of the present invention and others disclosed a newmethod in the course of 1997 (Picard G., Nevernov I., Alliata D. andPazdernick L., Langmuir, 13, 264 (1997)). The method has been markedwith the acronym DTLF (Dynamic Thin Laminar Flow), and comprises arotary cylinder that compresses a monolayer of soluble proteins. It wasspecifically planned in order to manufacture monolayers so solubleproteins, even if monolayers of soluble particles can also be easilyrealized. The features of this DTLF method are high-speed production,low amounts of materials being used, continuous production andpreparation of bidimensional crystals. The newly prepared monolayer canalso be deposited for further analysis on a solid, unmoving substrate.In other words, the device can be moved on a fixed substrate in order todeposit monolayers.

[0007] Even if in this study a machine has been disclosed that proved tobe functional with proteins, the basic principles governing the DTLFmethod were not explained. This means that the use of such apparatus canbe even useless if the basic forces are not controlled.

[0008] Object of the present invention is solving the above prior artproblems, providing the basic principles for the DTLF method in such away as to extend the application to the preparation of amorphous orcrystalline monolayers of all kind of particles and their followingtransfer on any type of liquid or solid substrate.

[0009] A further object of the present invention is providing anapparatus for the simple, efficient and inexpensive realization of theabove mentioned inventive method.

[0010] The basic principles of the DTLF method are based on thecombination of three different processes. The first one is that it isnecessary to use a thin liquid film: its thinness must be in themicrometer range. The second is the control of electrical charges of theparticles in the thin liquid film in order to provoke the adsorption ofparticles to the gas-liquid interface without provoking the adsorptionbetween them to the gas-liquid interface or in the liquid film itself.The third part is that, in order to create a force to drive particlesagainst an edge for compression, the surface, on which there is the thinliquid film, is moved. This movement pushes the thin liquid film aheadand creates, through the liquid viscosity, a surface force that finallypushes particles ahead.

[0011] The above and other objects and advantages of the invention,which will appear from the following description, will be obtained witha method as claimed in claim 1 and with an apparatus as claimed in claim11. Preferred embodiments and non-trivial modifications of the presentinvention are claimed in claims 2 to 10 and 12 to 21.

[0012] The present invention will be better described by some preferredembodiments thereof, provided as a non-limiting example, with referenceto the enclosed drawings, in which:

[0013]FIG. 1 is a side schematic view of a first embodiment of theapparatus according to the present invention;

[0014]FIG. 2 is a side schematic view of a second embodiment of theapparatus according to the present invention;

[0015]FIGS. 3 and 4 are optical microscopic images of particles on theupper part of the rotary element of FIGS. 1 and 2;

[0016]FIGS. 5 and 6 are optical microscopic images of small particlesrealized with the DTLF method of the present invention; and

[0017]FIG. 7 is an image from an atomic force microscope of a monolayerof ultrasmall particles realized with the DTLF method of the presentinvention.

[0018] In the course of the present description, the term “particle”means every type of molecules, polymers or aggregates whose meandiameter size is less than 100 microns.

Basic DTLF Method Principles

[0019] The DTLF method requires the simultaneous existence of twocharacteristics: a liquid subphase around 1 to 10 micron thick and onemobile surface. This thinness is important for the DTLF process becausethe particles in the thin liquid film will meet several times thegas-liquid interface during their transport due to the mobile solidsurface. Another important aspect of the DTLF process is that thethinness of the film means having to deal with very small liquidvolumes, in the microliter range. That means moreover that whichevermodification of the physico-chemical features of the liquid filmrequires injecting or pumping outside small amounts of buffers orsolutions. Moreover, the qualitative answer to any subphase modificationis fast.

[0020] The second important feature is that the surface, on which thethin liquid film rests, is moving. This movement drives the solid-liquidinterface and, because of the viscosity or the liquid, this movement istransmitted layer by layer up to the gas-liquid interface. Thesemovements provoke the convection in the thin liquid film that transportsparticles towards the gas-liquid interface in an efficient way.Moreover, this transport is eased by the Brownian motions when theparticles are at molecular level. Production efficiency can easily reach100%.

[0021] These two features have been described in the above-said priorpublication (Picard G., Nevernov I., Alliata D. and Pazdernick L.,Langmuir, 13, 264 (1997)). However, these features would be uselesswithout other considerations. The apparatus described in suchpublication, for example, would make particles endlessly rotate aroundthe cylinder, if no further manipulations of the thin liquid film werecarried out. These manipulations essentially comprise the adjustment ofsurface charge densities for the particles, that is an important factorin the determination of the adsorption of particles at the gas-liquidinterface.

[0022] Particle stability in the mass occurs because repulsion forcesbetween particles are greater that attraction forces. For example, ahigh surface charge density means that the particles will remain in themass, in solution or suspension. In this condition, no formation ofmonolayers is possible. The weakening of the surface charge density forparticles will also weaken the repulsion force. It has been determinedthat the first phenomenon that takes place is the adsorption ofparticles at the gas-liquid interface. The final result, which isassembling the particles in a monolayer, is the same. Essentially, withthe DTLF method the only two parameters to be controlled are the ionicforces in the subphase, for the particle A/W adsorption, and the surfaceforces that are pressing the particles onto the monolayer, the surfaceforce depending only on the cylinder rotation speed and the thin liquidfilm thickness. The further reduction of the repulsion forces provokesat the same time a second phenomenon that is the particle-particleadsorption at the gas-liquid interface. Therefore, aggregates areobserved on the liquid surface, while in the mass particles remainbalanced. Going on reducing the repulsion force between particles,particle to particle adsorption in the mass will be created. That willgenerate the precipitation of particles in the mass.

[0023] Clearly, the DTLF method works as soon as the particles insuspension or in solution are under unbalance conditions. In themajority of cases, this condition is present near the iso-electricpoint. A range of subphase conditions for every type of particles existfor an optimum adsorption at the gas-liquid interface. This optimumconditions can be found by injecting and pumping out liquids during themonolayer treatment, and following the monolayer production in realtime.

[0024] The fact that the surface, on which the thin liquid film rests,is moving implies that as soon as the particles are absorbed, they arecompressed against the edge of the growing monolayer. Particles arriveone after the other. This sequence of arrival is very favorable for theformation of large bidimensional crystals with particles. Such crystalshave been observed for protein and polystyrene particles. In principlethere are no limits for the size of particles and the nature of theirmaterial can be gold, silver, glass, etc.

[0025] The inventive apparatus

[0026] With reference to FIGS. 1 and 2, two preferred embodiments areshown of an apparatus to carry out the DTLF method according to thepresent invention. The inventive apparatus shown in FIG. 1 comprises arotary member 1, in this case a clockwise-rotating cylinder, to which aninjection module 8 is connected, this module 8 being equipped with threeopenings with respective inlet and cutlet channels for the fluid: achannel 9 through which a thin liquid film 2 is injected, throughadequate means (not shown), this film 2 containing a suspension ofparticles or proteins 3; a channel 10′ through which, through adequatemeans (not shown), adsorption reagents 10 are injected to be put incontact with particles 3 in suspension in the thin liquid film 2; and achannel 11 connected to a suction pump not shown) to suck the thinliquid film 2 after the deposition of the monolayer 5.

[0027] The apparatus in FIG. 1 comprises moreover a substrate 7 on whichthe monolayer 5 is deposited. According to what is shown in FIG. 1,particles 3, after their surface charge density is modified by means ofcontact with reagents 10, are carried to the surface, that is at thegas-liquid interface, and are therefore absorbed, as clearly appearsfrom the particles designated by reference number 4. The rotation of therotating member (arrow A) pushes particles 4 one against the other toform a continuous and uniform monolayer 5. By going on rotating member 1and parallely by moving it in the lengthwise direction to a substrate 7(in FIG. 1 in the direction of arrow B that is the opposite regardingthe rotation sense for member 1), the monolayer 6 is deposited on thesubstrate 7 together with the thin liquid film 2, that is then suckedaway by means of suction means 11. A monolayer of particles 6 will beobtained and therefore transferred onto the solid substrate 7, than incase of FIG. 1 is a hydrophobic substrate made with a slide of glass ormetal.

[0028] The embodiment in FIG. 2 is the same as the one in FIG. 1 (andtherefore the same parts are designated by the same reference numbers),apart from for the fact that the substrate 7 is made of hydrophylicmaterial, that is composed of a clean glass plate or a mica sheet.Therefore, in this case, the deposition of monolayer 6 on it is carriedout making the rotary member 1 advance in the direction of arrow C, thatis the same as the rotation sense of the member.

[0029] The apparatus and method of the present invention operate in asimilarly effective way if the substrate 7 is composed of any type ofliquid, on which it will therefore be possible to deposit one or moremonolayers (through one or more successive application passes) ofparticles or molecules.

[0030] Some practical applications of the method and the apparatusaccording to the present invention provide that such adsorption reagents10 are made of an acidic solution to a pH equal to approximately 4.0 forparticles 2 of polystyrene or protein molecules. According to anotherexample, the adsorption reagents 10 are composed of a solution made of70% of acetonitrile for particles 2 of carbon 60 in a toluene film.According to a further example, the adsorption reagents 10 can be asalts solution, in particular a cadmium sulfate solution for molecules 2or proteins of the holoferritin type.

[0031] The invention can be practiced by realizing filters forultrafiltration, whose pore diameter ranges from 1,000 to 1 nanometer.

EXPERIMENTAL EXAMPLES

[0032] The DTLF method will be described hereinbelow with reference to aset of experimental tests, whose results are shown in FIGS. 3 to 7. Allexperiments were performed in a clean room. The dust level was measuredby a commercial dust detector for white room quality control.

[0033] The prototype testing this new method is shown in FIG. 1, in sideand top views. The glass cylinder was 6 mm in diameter and 50 mm long.The glass cylinder surface was polished with fine abrasives forcommercial lenses until no scratch could be seen at 1000× magnificationwith an optical microscope. A hemi-cylindrical trough was obtained bycutting out and drilling a 10×3.5× 0.5 cm PTFE plate. A DC electricmotor with a speed control up to 3 Hz was used to drive the glasscylinder. DC electric motor and glass cylinder were mechanicallyconnected by means of a thick rubber tube, in order to transmit torquewhile damping vibrations. The cylinder was held horizontally by two PTFEcircular plates drilled at 2 mm from the center. The cap between thecylinder and the hemi-cylindrical trough could be adjusted to about 300μm by simply rotating the circular plates. After a vertical position wasfound, the circular plates were clamped firmly on a rigid Plexiglassstructure.

[0034] The polystyrene particles for the tests were from InterfacialDynamics Corporation (IDC), Portland, Oreg., U.S.A. The particleconcentration was always 4% w/w. Only the fluorescent 0.22-μm particleswere from Polysciences, Warrington, Pa. The buffers and NaCl (99.99%)were from Merck Water was distilled (Aquatron BS I) and demineralized(Elgastat UHQ II) before use. Its surface tension was higher than 72mN.m⁻¹ and its conductivity equal to 18 MΩ.cm⁻¹.

[0035] Six channels controlling subphase volume inlet, pH and thinliquid film thickness above the cylinder were drilled in the lateralportions of the PTFE hemi-cylindrical trough. At the bottom of thetrough and parallel to the cylinder axis a groove was drilled tohydraulically connect all channels for a better mixing of infectedfluids. When salt was used for the formation of the latex particlemonolayer, the subphase was thoroughly rinsed by simultaneouslyinjecting pure water and pumping the subphase away. The prototype wasmounted on an optical microscope bench for in situ observation of theparticle monolayers and the rinsing procedures.

[0036] The subphase pH or salt concentration were gradually modified andadjusted with the syringe graduations. The thickness of the thin liquidfilm around the cylinder was finely controlled by the use of an opticalmicroscope focal depth. The subphase conditions are shown in Table 1with the corresponding observations.

[0037] The preparation of 6- to 0.6-μm particle monolayers was quiteeasy, because the monolayers could be seen as a white coating materialover the cylinder. Thus the procedure just consisted in injecting theparticles into the thin liquid film with a pipette and in looking at thegrowing white film. The injection of particles was gradual, keeping themonolayer speed of preparation constant. Once the hemi-cylindricalsurface was totally covered, the injection was stopped. The film couldbe observed during the growing process, and transferred later onto asolid substrate for further observations. The reduction of the particlesize made the direct visual observation of the monolayer increasinglydifficult. The white film became whitish with the 1-μm spheres andtranslucide with the 250 nm ones. At this point the observationprocedure was made by means of an optical microscope.

[0038] For particles smaller than 250 nm an optical microscope with itsobjective over the top of the cylinder, in incident light and darkfieldmode illumination, was used. The in situ observation of the particlemonolayers required an interval of time between the particle injectionand the setting up of the microscope, because of the shortobjective-water distance of about 1 mm. Photographs were taken after thetransfer on a solid substrate with a 100 ASA commercial film.

[0039] When salt was used, a rinsing subphase was used. A microscope at200× magnification vertically positioned above the cylinder was used forthe observation of polystyrene monolayer preparations. With the opticalmicroscope focused on the film, the cleaning sequence started. Thisallowed the liquid to axially flow axially from one extremity of thecylinder base to the other. 4- to 16-ml of pure water was usually flownthrough.

[0040] The method of transferring the particle monolayer was thehorizontal deposition. It consisted in bringing in contact a water filmon a wetted hydrophilic surface with the water film around the rotatingcylinder. The surface concerned was a hydrophilic microscope cover slipwith its dry surface on the PTFE surface and its upper surface wetted.The contact was made at the base of the cylinder by sliding horizontallythe microscope cover slip until the two water films fused by capillaryforces. As soon as the contact was made the particle monolayer over thecylindrical thin film surface, blocked until then by the hydrophobicPTFE corner, was driven forward by the cylinder over the wetted glasssurface.

[0041] After this horizontal movement, the excess liquid that connectedthe cylinder and the microscope cover slip was pumped away by using the2-way pump, and the particle monolayer was finally disconnected from thecylinder. The particle monolayer was then left free floating over a thinliquid film, which was coating the glass surface. The thin liquid filmevaporated through the particle monolayer, thus the particle monolayermade a slow descent until the touch down onto the microscope cover slip,that is a particle monolayer was transferred by horizontal depositionover the glass slide.

Dynamics of Thin Liquid Films

[0042] A glass cylinder revolving under a particle monolayer with a thinfilm of water in between can be represented by two concentric cylinders,the central one rotating and the outer one fixed, with fluid in between.This is the well-known Couette viscometer. The mathematics connectingthis viscometer with the DTLF method has already been developed for theexperiments with proteins. Although the experimental conditions werewith polystyrene spheres, the Taylor number remains Ta= 1.48×10⁻⁵. Sinceinstability appears at the critical Taylor number Ta_(c)=1.712, thiscertifies that the flow between the particle monolayer and the glasscylinder surface will be laminar. The thinness of the liquid film islargely responsible for this high stability.

[0043] The particle monolayer dimensions being in a 10,000:1 ratio withthe film thickness, the end effects can be neglected and the cylindercan be considered as infinite. Again, using the same argument, thecylinder radius being in a 1,000:1 ratio with the film thickness, thecylinder-in-cylinder model can be mathematically reduced to a plateparallely moving above a fixed one, with a fluid in between. In oursystem the moving plate was the cylinder surface and the fixed plate,the particle monolayer. The force created by the rotating cylinder onthe particle monolayer was put in equation:$\Pi = {{\frac{2{\pi \cdot \eta \cdot R_{C} \cdot f \cdot x}}{z}\quad 0} \leq x \leq L_{h}}$

[0044] where η is the liquid viscosity (for water, 10⁻³ kg/m s; R_(c)the cylinder radius, f the cylinder rotation frequency, x the length ofthe monolayer and z the thickness of the thin liquid film. L_(h) is thehemi-cylindrical circumference. It was demonstrated that you can reachsurface pressures up to collapse point.

[0045] Optical microscopy at the A/W interface

[0046] The injection of 6-μm polystyrene spheres in the thin liquid filmof water produced a growing white solid film of polystyrene particles.At neutral pH, the electric charges around the particles createdrepulsive forces that efficiently provided any kind of particleadsorption. As a consequence, the particles turned with the cylinderwithout making monolayers. Lowering the pH to 4.0 reduced the surfacecharges of the particles. Then, a progressive adsorption of particles atthe A/W interface occurred. The regular rotation of the cylindercompressed the just-emerged particles, leading to a uniform monolayerpreparation. At pH 4.0, the ionic repulsive forces are still enough tokeep the particles apart at the A/W interface. This repulsion is wellknown to be important for the formation of 2D crystals. As a matter offact 2D crystals were observed, in particular when the injection wassmoother see FIG. 3). Further reducing the electric charges of theparticles by lowering the pH to 3.5 led to surface aggregation. In fact2D fractals were observed (see FIG. 4). At pH 3.0, 3D aggregates wereclearly visible. This indicates that particle-particle adsorption in thebulk occurred rapidly.

[0047] This dependence of monolayer preparations on the subphase ionicconditions proved to be true for all sulfate- or carboxyl-coatedlatexes; however, the values varied somewhat from size to size (seeTable 1). This observation will be further analyzed in the Discussionsection. Another relevant observation is that reducing the pH to 0.0,did not provoke the aggregation of the 0.250 μm CML particles, while anamount of 1 M of salt in pure water provoked their absorption at the A/Winterface. Increasing the salt concentration, which is screeningfurthermore the surface charges of the particles, led to surface andbulk aggregations, as well.

[0048] Optical microscopy at the air-solid interface.

[0049] The monolayers at the A/W interface could be cried down onto thecylinder glass surface, and examined with the optical microscope.Otherwise the film could be transferred onto a glass slide or a micasheet. In any cases the observation at the air-solid interface allowed abetween evaluation of the particle packing.

[0050]FIG. 5 shows the 1.17-μm particles that were assembled by DTLFmethod. It is quite clear that the good packing of particles occurredbecause of the introduction of the particles in the monolayer one afterthe other. FIG. 6 shows smaller particles, i.e. 0.614 μm, which werealso well packed. It must be noted that the linear speed of preparationof 0.614-μm particle monolayer could reach 1 mm/s.

[0051] AFM microscopy at the air-solid interface.

[0052] The nanospheres were seen with the AFM microscope. In FIG. 7, anAFM image of a 127-nm particle monolayer is shown. In this image, theparticle monolayer is quite uniform. It is also possible to see smallnanosohere 2D crystals. Also in this case, the linear preparation speedof the monolayer was ≈1 mm/s.

[0053] Table 1 summarizes the results. The data are shown in decreasingorder of particle diameter. Hydro-affinity was the only parameter thatprevailed for the choice of the ionic condition. One of the mainfeatures is that the subphase conditions for the preparation of particlemonolayers were not much different between the biggest and the smallestspheres. TABLE 1 particle properties, subphase initial conditions andobservation of the monolayer preparations. Visual Diam- Surface aspectVisual eter Particle charge on aspect (μm) Time(s) Type (μC/cm²) [NaCl]water on solid Microscopic (1) (2) (3) (4) M pH (5) (5) observation6.240 — Sulfate 4 Nil 7.0 — — Nothing ″  30 ″ ″ ″ 4.0 Mat Mat 2D whitewhite crystals ″ — ″ ″ ″ 3.5 Frosty Frosty 2D aggregates ″ — ″ ″ ″ 3.0Granular Granular 3D aggregates 1.170  30 Carboxyl 19 ″ 4.0 WhitishColorful 2D (6) crystals 1.010 — CML 923 ″ 4.0- — — Nothing 3.0 0.833 ″CML 1135 1.0 7.0 ″ ″ ″ 0.614 ″ Carboxyl 16 Nil 4.0 ″ ″ ″ 0.250 — CML 226″ 4.0- — — Nothing 0.0 ″ — ″ ″ 2.5- 7.0 Grainy Grainy 3D 1.5 white whiteaggregates ″ <30 ″ ″ 1.0 ″ Transparent Bluish 2D crystals 0.220 ″Carboxy Unknown 1.5 ″ ″ ″ 2D * YG crystals 0.127 ″ Carboxyl 4 1.0 7.0 ″Blue 2D crystals ″ ″ ″ Nil 9.0- ″ ″ 2D 8.0 crystals ″ — ″ ″ 7.0- Barely″ 2D 4.0 visible aggregates ″ — ″ ″ 3.5- Whitish ″ 3D 3.0 aggregates0.053 — ″ 0.2 ″ 5.0 — — Nothing ″ <30 ″ ″ 4.0 Transparent Blue 2Dcrystals

[0054] Notes

[0055]¹ Estimated by the IDC company using a TEM. An average was madewith 500 particles chosen at random. However, our estimation based onthe microscopic observation of particle 2D arrays and using a standardgrid for calibration indicated that the particles were slightly bigger.

[0056]² These times are the shortest measured with the minimum particleamount to make a monolayer. For 2D array preparations the linear speedof the monolayer preparation was slowed down in order to increase the 2Darray proportions. With nanoparticles an optical microscope was used.However, in spite of this approximation, the values are clearly givingan idea concerning the mechanisms acting in the DTLF method.

[0057]³ The polystyrene particle or properties depend on the surfacefunctional groups that were created. A relevant parameter is the CCC,for hydrophobic latexes it is either at 0.25 M univalent ionconcentration at pH 7.0, or at pH lower than 4.0. for the CML it is aunivalent ion concentration higher than 1.0 M at pH 7.0.

[0058]⁴ The surface charge concentration, provided by the producer, isfor polystyrene spheres in pure water.

[0059]⁵ The visual aspect as seen directly from 6.24 μm down to 0.250μm. At smaller sizes, the procedure followed has already been described.

[0060]⁶ The colors appeared by light interference. In the sunlight,bright rainbow colors could be seen by tilting the particle monolayer.Each color covered alternatively and uniformly the whole surface or themonolayer according of the angle of tilt. This simple observationindicates the homogeneity of the film itself.

[0061] * This is the only particle coming from a different producer. Itsconcentration was 2.2% w/w. Its surface was not characterized, howeverthe production of a particle monolayer could be easily realized.

Discussion

[0062] The particle monolayer growth was visible to the naked eye downto 250 nm. It was regulated by the particle injection and could beadjusted so as to be a constant: the response to the injection was animmediate monolayer growth, upon stopping the injection the monolayerspeed of preparation gradually stopped within a minute. The growingmonolayer reduced the available free A/W interface for particleadsorption, however this reduction seemed not to have affected the speedof preparation, which remained steady. These two observations bythemselves already indicate that convection in the thin liquid filmunder the free A/W interface was the leading process for micro- tonanoparticles down to 250 nm. For monolayer preparations with smallerparticles the speed of preparation could not be adjusted, instead aninjection was made and a particle monolayer was observed in situ withthe optical microscope like in the case of proteins. However, the timeof preparation was for them also (see Table 1) very short ≈30 s). Thissuggests that for particles down to 55 nm the Brownian motion was stilldominated by convection.

[0063] The physico-chemistry of the DTLF method is based on the surfaceelectric charges and their control by pH or salt concentration. It isimportant to know that the surface charges provided in Table 1 are theresult of a computation based on the stiochiometric charges contents.For carboxil- and sulphate-coated particles, the computation whollycorresponds to reality. However, for CML particles, the majority ofcharges are buried, leaving a surface charge density comparable with theother types. This volume distribution makes CML particles behaveinterestingly. Taking the 0.833-μm CML spheres as an example, thesurface electric charge was 1135 μC/cm². The increasing of the subphaseacidity up to pH 0.0 could not create the particle adsorption at the A/Winterface. For the 1.01-μm. CML spheres with a surface charge of 923μC/cm², the preparation of the particles monolayer occurred at asubphase pH of 2.0. For the 0.250-μm CML spheres with a surface chargeof 226 μC/cm², the same conditions as the highly charged 0.833-μmspheres prevailed. For the carboxyl particles with surface charges lessthan 20 μC/cm², simply increasing the subphase acidity to PH 4.0 wasenough to provoke the surface adsorption without creating the formationof 2D aggregates. In the case of the 0.127-μm particles, the subphase pHcould be basic at pH 9.0. In two cases, monolayers of highly chargedparticles could not be prepared by only increasing the subphase acidity,however, screening the surface charges with salt addition proved to bevery efficient. On the contrary all the particles wish the weakestsurface charges subphase mild acidic conditions at pH 4.0 weresufficient.

[0064] It may be surprising that for the 0.127-μm particles in thesubphase at pH 9.0, a particle monolayer was prepared. This resultescapes the theoretical frame, as the adsorption between the particleand the A/W interfaces should not have occurred. It seems that the0.127-μm particle behavior changed from the macroscopic to thenanoscopic scale. As a matter of fact, their observation to the nakedeye on the water surface became impossible. However using the opticalmicroscope, like in the case of proteins, the monolayer could beobserved. The injection of particles yielded a particle monolayer suiterapidly, in a few words their behavior was nearly the one of asurfactant, as there was no formation of aggregate in the bulk. Moreoverat subphase pH below 4.0 the particles adsorbed at the A/W interface andmade 2D aggregates. Lowering more the pH provoked the aggregation in thebulk. These two behaviors were observed in the case of the othercarboxyl particles.

[0065] The experimental data for the time of preparation were comparedwith the two mathematical models based on Brownian and mechanicalconvection models (see Table 2). TABLE 2 Experimental and simulatedtimes for A/W interface full coverage with microspheres. Particle Timefor A/W interface full coverage (s) Diameter Experimental⁽¹⁾ Numericalsimulation (μm) DTLF Brownian⁽²⁾ Convect⁽²⁾ 6.240 ≈30 1646 34 1.170 ″554 27 1.010 ″ 512 27 0.833 ″ 458 26 0.614 ″ 400 27 0.250 <30 230 24

[0066] The concentration used in the models is given by the producer,i.e. 4% w/w, injected in the 25-μL meniscus. It must be noted that thetime of experimentation is quite approximate. The particle injection wasrelatively slow, in order to smooth the particle monolayer preparation.For the 6.24-μm particles down to the 250-nm size, the convection in thethin liquid film was clearly the mechanism by which the particlesreached the A/W interface. The Brownian motion alone would have been byfar too slow.

[0067] However, to better evaluate the potential of the DTLF method,that is the preparation of crystalline monolayers of particles ormonolayers in continuous mode, a realistic situation could be that thetriple line air/water/monolayer is somewhere between both menisci.Practically, it would be better that this triple line were at the tophalf circumference prior the transfer, for example, on a solidsubstrate. Here a rotation frequency of 1 Hz and a 5 μm thin liquid filmcan be assumed. Given these parameters, for particles or otherultra-fine particles smaller than 3 nm, the Brownian motion becomes moreefficient than the mechanical convective motions to bring particles tothe A/W interface. In other words the DTLF method moves big particlesmechanically to the A/W interface, while the ultra-small particles willself-diffuse to the water surface. In both cases the final result, whichis assembling the particles in a monolayer, is the same. Essentiallywith the DTLF method the two only parameters to be controlled are theionic force in the subphase, for the particle A/W adsorption, and thesurface force that is pressing the particles onto the monolayer, thesurface force being only dependent of the cylinder rotation speed andthe thin liquid film thickness.

[0068] The particle monolayer preparations produced colorful effects.For one monolayer thick, these effects were most spectacular withparticles having about 0.8-μm diameter. According to a recentpublication, interferences due to an optical light paths differencemade, by the polystyrene refractive index, could explain theobservations. A simple mathematical model based on the reflection of thelight on the top of the monolayer and on the air-glass interface belowthe monolayer, combined with the difference of optical path, fitted verywell the experimental data. However in our work a simple experiment wasperformed to further test this mathematical model. The monolayer washeated mildly at 90° C. for a series of short times, and observed withthe optical microscope. The colors disappeared at the very same momentwhere the spherules started to soften and fuse, giving a smooth-bumpedmonolayer surface It must be noted that this mild heating did not changethe optical path. Thus the mathematical model reported has to berefined, as the film thickness parameter cannot explain the results,this problem will be analyzed in the future.

[0069] Another interesting observation came from the above publicationby Picard et al. disclosing the DTLF method with proteins. A monolayerwas efficiently prepared, in spite of the fact that the protein used washighly hydrophilic. It was dissolved in water with some glycerol topermit its storage at low temperature. Glycerol kept this protein fromcrystallization and its removal triggered crystallization as theaddition of salt does in the subphase for particles. Combined with thecontrol of subphase pH, 2D crystallites with DTLF method were produced.The preparation of protein monolayers is more difficult than withparticles, due to self-crystallization, however, the experiments withparticles indicated that the basic principles of the DTLF method werequite well understood.

1. Method for the preparation of monolayers of particles or molecules(3), said method including the following steps: injecting a thin liquidfilm (2) containing said particles or molecules (3) dispersed therein onan external surface of a rotary member (1); adjusting a surface chargesdensity of said particles or molecules (3) through the injection ofadsorption reagents (10), said step of adjusting the charge densitycarrying said particles or molecules (3) at a surface of said thinliquid film (2); carrying said particles or molecules (3) adsorbed at agas-liquid interface of the thin liquid film (2) into a uniformmonolayer (5); transferring said monolayer (5) from the surface of saidthin liquid film (2) to the solid substrate (7); and making saidrotating element (1) move in a longitudinal direction relatively to saidsubstrate (7), said monolayer (5) separating from said thin liquid film(2) and adsorbing to said substrate (7).
 2. Method according to claim 1, characterized in that said substrate (7), is hydrophilic, said liquidfilm (2) being also attached to said substrate (7), said longitudinalmovement step of said rotary member (1) being carried out in the samedirection as the rotation direction of said rotary member (1), saidmethod further comprising the step of sucking said liquid film (2)attached to said substrate (7) away from said substrate (7).
 3. Methodaccording to claim 2 , characterized in that said substrate (7) iscomposed of a clean glass plate or a mica sheet.
 4. Method according toclaim 1 , characterized in that said substrate (7) is hydrophobic, saidliquid film (2) not being attached to said substrate (7) but remamainingon the external surface at said rotary member (1), said longitudinalmovement step of said rotary member (1) being carried out in thedirection opposite to the rotation director of said rotary member (1),said method further comprising the step of sucking said liquid film (2)away from said substrate (7).
 5. Method according to claim 4 ,characterized in that said substrate (7) is composed of a glass or metalplate.
 6. Method according to any one of the previous claims,characterized in that said adsorption reagents (10) are composed of anacid solution at a pH equal to 4.0 for particles (2) of polystyrene orprotein molecules.
 7. Method according to any one of the previousclaims, characterized in that said adsorption reagents (10) are composedof a 70% acetonitrile solution for particles (2) of carbon 60 in atoluene film.
 8. Method according to any one of claims 1 to 5 ,characterized in that said adsorption reagents (10) are a saltssolution.
 9. Method according to claim 8 , characterized in that saidsalts solution is a cadmium sulfate solution for molecules (2) ofproteins of the holoferritin type.
 10. Method according to any one ofthe previous claims, characterized in that said thin liquid film (2) hasa thickness of the order of microns.
 11. Apparatus for the preparationof monolayers of particles or molecules (3), said apparatussubstantially comprising: means for injecting a thin liquid film (2)containing said particles or molecules (3) dispersed therein on anexternal surface of a rotary member (1); means for adjusting the surfacecharge density of said particles or molecules (3) through the injectionof adsorption reagents (10), said means for adjusting the charge densitycarrying said particles or molecules (3) on said surface of said film ofliquid (2); means to keep said rotary member (1) rotated in order todrag said particles or molecules (3) placed on said surface of saidliquid film (2) so that they form a substantially uniform monolayer (5),said rotary member (1) being actuated so as to put said monolayer (5) onsaid surface of said liquid film (2) in contact with a substrate (7);and means for advancing said rotary member (1) in a longitudinaldirection with respect to said substrate (7), said monolayer (5) beingdetached from said liquid film (2) and being attached to said substrate(7).
 12. Apparatus according to claim 11 , characterized in that saidsubstrate (7) is hydrophylic, said liquid film (2) being also attachedto said substrate (7), said means for longitudinally advancing saidrotary member (1) carrying out said advancement in the same direction asthe rotation direction of said rotary member (1), said apparatus furthercomprising means (11) for sucking said liquid film (2) attached to saidsubstrate (7) away from said substrate (7).
 13. Apparatus according toclaim 12 , characterized in that said substrate (7) is composed of aclean glass plate or a mica sheet.
 14. Apparatus according to claim 11 ,characterized in that said substrate (7) is hydrophobic, said liquidfilm (2) not being attached to said substrate (7) but remaining on theexternal surface of said rotary member (1), said means forlongitudinally advancing said rotary member (1) carrying out saidadvancement in an opposite direction to the direction rotation of saidrotary member (1), said apparatus further comprising means (11) forsucking said liquid film (2) away from said substrate (7).
 15. Apparatusaccording to claim 14 , characterized in that said substrate (7) is madeof a glass or metal plate.
 16. Apparatus according to claim 11 ,characterized in that said substrate (7) is composed of a liquid. 17.Apparatus according to any one of claims 11 to 16 , characterized inthat said adsorption reagents (10) are an acid solution at a pH equal to4.0 for particles (2) of polystyrene of protein molecules.
 18. Apparatusaccording to any one of claims 11 to 17 , characterized in that saidadsorption reagents (10) are composed of a 70% acetonitrile solution forparticles (2) of carbon 60 in a toluene film.
 19. Apparatus according toany one of claims 11 to 15 , characterized in that said adsorptionreagents (10) are a salts solution.
 20. Apparatus according to claim 19, characterized in that said salts solution is a cadmium sulfatesolution for molecules (2) of proteins of the holoferritin type. 21.Apparatus according to any one of claims 11 to 16 , characterized inthat said thin liquid film (2) has a thickness of the order of microns.